Plants synthesise a diverse range of natural products. Many of these compounds are specialised metabolites that are produced only by certain taxonomic groups (1). Plant-derived natural products have important ecological functions, often serving as attractants or deterrents in interactions with other organisms (1,2). The ability to synthesise particular natural products is therefore likely to be a consequence of niche colonisation and adaptive evolution (2,3). Currently we know very little about how new metabolic pathways arise. A better understanding of the origin and nature of the genes and enzymes that comprise natural product pathways will enable us to probe the mechanisms underpinning the generation of metabolic diversity.
Avenacins are antimicrobial triterpene glycosides (saponins) that accumulate in the roots of oats (Avena spp.) (4,5). The ability to synthesise avenacins is restricted to members of the genus Avena (4) and has arisen relatively recently—since the divergence of oats from other cereals and grasses (6). The major avenacin, A-1, contains the fluorophore N-methyl anthranilic acid and so confers a bright blue fluorescence on the roots of oat seedlings under ultraviolet illumination. In previous work we have exploited this fluorescence as a screen to isolate saponin-deficient (sad) mutants of diploid oat (Avena strigosa) following chemical (sodium azide) mutagenesis (5). sad mutants are compromised in disease resistance to a range of fungal pathogens, demonstrating that avenacins confer broad-spectrum protection against microbial attack (5). These experiments have provided the first direct evidence for a role for preformed antimicrobial compounds in plant defence.
Avenacins are synthesised from the isoprenoid pathway and share a common biogenetic origin with sterols, the two pathways diverging after 2,3-oxidosqualene (FIG. 1) (4, 7-9). In primary sterol biosynthesis 2,3-oxidosqualene is cyclised to cycloartenol by cycloartenol synthase. Cycloartenol is then converted to other sterols via a series of intermediates that includes obtusifoliol. The first committed step in the avenacin pathway is the cyclisation of 2,3-oxidosqualene to the triterpene precursor β-amyrin, catalysed by the oxidosqualene cyclase enzyme β-amyrin synthase (7-9). β-Amyrin is not antimicrobial but is converted to the biologically active avenacins by a series of uncharacterised modifications that are predicted to involve oxidation, glycosylation and acylation (9).
From genetic analysis of our mutant collection we originally defined eight loci for avenacin synthesis (Sad1-8).
We have previously cloned Sad1, the gene encoding β-amyrin synthase (FIG. 1) (8), (and see Haralampidis et al., PNAS Vol. 98, No 23, pp 13431-13436, Nov. 6, 2001; see also WO01/46391), but have not previously reported the sequence of the functional promoter of this gene. Our data indicate that Sad1 is likely to have been recruited from sterol metabolism by duplication and divergence of a plant cycloartenol-synthase like gene and that this is a relatively recent evolutionary event (6,8). Remarkably, six of the seven other Sad loci that we have defined by mutation (Sad-2,3,5,6,7 and 8) co-segregate with Sad1, indicating that the genes for avenacin biosynthesis are clustered (5,6). Although many examples of clustered genes for natural product pathways have been reported in microbes, gene clusters of this kind are not a common phenomenon in plants (2,6). The reason for clustering of avenacin biosynthetic genes is not yet known.
WO2006/044508, (see also Qi et al., PNAS, Vol. 101, No. 21, pp. 8233-8238, May 25, 2004) relate to the cloning of the Sad2 gene, although limited information was provided about the function and specificity of the promoter of that gene.
The CYP51 sterol demethylases are regarded as the most ancient cytochrome P450 family. They are highly conserved across the animal, fungal and plant kingdoms and are only known to have a single strictly conserved function—in the synthesis of essential sterols (10-13). AsCYP51H10 belongs to a new subfamily of divergent plant CYP51 enzymes (CYP51H) that until now has been defined only by rice sequences of unknown function (11). This subfamily is not represented in Arabidopsis or other dicots. Our data indicate that AsCYP51H10 has undergone neofunctionalisation and is required for the synthesis of defence-related antimicrobial triterpene glycosides (avenacins) but is dispensable for primary sterol biosynthesis. To our knowledge this is the first report of a CYP51 enzyme that has acquired a new function. Our demonstration that both Sad1 (6, 8) and Sad2 (AsCyp51H10) have been recruited from plant primary sterol metabolism indicates an intimate evolutionary connection between the sterol and avenacin pathways. However the expression patterns of Sad1 and Sad2 have been refined. While their sterol biosynthesis counterparts (the cycloartenol synthase and obtusifoliol 14α-demethylase genes, respectively) are expressed constitutively throughout the plant, expression of Sad1 and Sad2 (which are 70 kb apart) is tightly regulated and is restricted to the epidermal cells of the root tip, the site of accumulation of avenacins.
The promoters from genes which are tissue specific (e.g. root, or root-tip specific) have utility inter alia in expressing transgenes in this manner. Thus it can be seen that the characterisation of the sequences and specificity of such promoters provides a contribution to the art.